Peripheral tolerance, or the elimination or functional silencing of potentially auto-reactive T cells, relies on the presentation of self-antigen to T cells. To understand how self-antigen presentation leads to tolerance or autoimmunity is the long-term goal of our work. This proposal is based on the hypothesis that lymph node stromal cells (LNSCs) are highly specialized antigen presenting cells that impose self-tolerance on circulating T cells. Several findings lead to this hypothesis. First, like medullary thymic epithelial cells (mTECs), LNSCs constitutively express peripheral tissue antigens (PTAs), and the autoimmune regulator (Aire) gene, a transcriptional regulator that partially controls PTA expression and central tolerance induction by mTECs. Second, LNSCs are localized in the cortex of the lymph node, which is an ideal location for interacting with circulating T cells. Third, LNSCs can process endogenously expressed antigen into peptide- MHC complexes and induce proliferation in naive, antigen-specific T cells. Fourth, the presentation of endogenously expressed PTA by LNSCs is sufficient to promote deletional tolerance among antigen-specific T cells. The objective of this proposal is to dissect the molecular and cellular mechanisms by which LNSCs present self-antigens to CDS and CD4 T cells and to evaluate their role in imposing tolerance to key autoantigens. The specific aims are to: 1) Investigate the general functional properties of mouse and human LNSCs. Here we will characterize the transcriptional profile of LNSCs and the role of Aire in these novel APCs. We will then assess whether PTA expression by mouse LNSCs is linked to disease susceptibility in experimental autoimmune encephalomyelitis (EAE) and type-1 diabetes (T1D);2) Define the role of LNSCs in promoting tolerance among MHC class I- and class ll-restricted self-reactive T cells. We will determine whether LNSCs promote tolerance to islet-specific glucose-6-phosphatase, a pancreatic antigen that is targeted in T1D using the 8.3 TCR transgenic mouse model in which pathogenic CD8+ T cells destroy insulin-producing beta cells. Conclusions from these studies will be tested for generality using the 5B6 TCR transgenic mouse model of EAE in which CD4+ T cells recognize the myelin protein, proteolipid protein;3) Ascertain whether the function of LNSCs is influenced by inflammation. We will examine the impact of inflammation on the function and gene expression profile of LNSCs. Results from these studies will elucidate whether LNSCs are constitutively tolerogenic or whether their capacity for self-antigen presentation is modified under inflammatory conditions.